In 2004, the United States passed legislation that made hundreds of researchers around the country guilty of a crime with consequences of up to $2,000,000 fines and a prison sentence of 25 years to life. What was that crime? Working with vaccinia—a live virus vaccine that essentially eradicated smallpox by 1980 and became the most widely used human immunization. While routine vaccination with vaccinia is no longer recommended, the virus is still researched in numerous laboratories and is even administered to those at risk for exposure to viruses in the smallpox family, such as laboratory workers, military, selected health-care workers, and first responders.[1] Certainly, legislators of the Intelligence Reform and Terrorism Prevention Act (IRTPA) didn’t actually intend to outlaw the vaccine that not only eliminated one of the world’s most feared diseases but continued to be useful in producing novel vaccines today.

What the IRTPA actually intended to do was to combat the national threat of three potentially harmful weapons—handheld surface-to-air missiles, or “MANPADS,” radiological dispersal devices, also known as “dirty bombs,” and the variola major virus, the causative agent of smallpox disease.[2] The problem was that in defining variola virus, legislators naively declared that “any derivative of the variola major virus that contains more than 85 percent of the gene sequence” would be illegal to produce, engineer, synthesize, or threaten to use.[2] The variola virus can be modified slightly while still maintaining its viral, dangerous nature, and thus limiting sequence similarity seems at first like a decent solution. But the problem is that within the bounds of an 85% modification also resides the sequence for vaccinia, a lifesaving vaccine against smallpox and a model system for studying the field of virology.

This legislative blunder may have resulted from plain oversight and carelessness, or a fundamental lack of understanding and knowledge about the field of biotechnology. Advances in the last four decades have brought smallpox and other pathogenic genetic sequences into the spotlight as potential biosecurity risks. But what exactly are these microscopic viruses made of that could possibly put them in the same category as missiles and radioactive bombs?

Parasitic Packages

Viruses, like the variola major virus that causes smallpox or the vaccinia virus that vaccinates people against it, are tiny, parasitic protein packages containing a molecule of genetic material. Viruses are one of the world’s biggest moochers (well, they’re actually microscopic, but don’t let their size fool you). On their own they are helpless, unable to produce energy or replicate themselves. However, as soon as they find a host cell, they can latch on, plunge their DNA or RNA contents into the cell, and hijack the cell’s machinery and nutrients in order to replicate themselves.[3]

Even if you are not a molecular biologist, you’ve probably heard of DNA, or deoxyribonucleic acid. Maybe you’ve just been told in high school biology that the “building blocks of DNA” make up the “genetic blueprint” of nearly all organisms, or maybe your uncle even had his genome—his complete set of DNA—sequenced by 23andMe hoping to find distant relatives sharing his Norwegian heritage.

DNA is made up of two strands of molecules called nucleotides, which are organized into pairs and wound up into a spiral staircase-like structure called a double helix. Each nucleotide is a chemical conglomeration of simple parts—a sugar molecule, a phosphate group, and one of four nitrogen-containing molecules called “bases” that define the sequence of DNA. These bases are identified by the letters C, G, A, or T. RNA, or ribonucleic acid, is a DNA-like molecule that is usually single stranded and has U building blocks in the place of Ts.[4]

As the fundamental ingredient of life, DNA has been extensively studied since it was identified in the late 1860s by Swiss chemist Friedrich Miesche and then characterized as a double helix by American biologist James Watson, English physicist Francis Crick, and English chemist Rosalind Franklin almost a century later.[4] The newfound understanding of the impressively potent, yet beautifully simplistic, model of DNA laid the foundation for many new fields and pursuits, including synthetic biology.

For over 35 years, scientists have been capable of performing de novo, or “from scratch,” synthesis of genetic material using a variety of methods like DNA recombination. Recombination takes pre-existing, template sequences and uses a “cut and paste” technique to create novel DNA molecules. A time consuming and difficult task, it took years to synthesize just one hundred base pairs of a desired gene and was only accessible to experts or researchers with appropriate laboratory setups.[5]

Today, advancements in chemical synthesis, engineering and computation have been leveraged to streamline the design, manipulation and creation of DNA. Like the world’s most accurate and direct language translator, DNA synthesizing machines take in the genetic sequence of Cs, Gs, As, and Ts (or Us), and then carry out chemical reactions to assemble the series of phosphates, sugars, and nitrogenous bases corresponding to the sequence input. Companies like DNA2.0, GenScript, and Twist offer this service for sequences of anywhere from a few hundred base pairs to several thousand base pairs.[6] Today, it is fast and affordable—the price per base pair is around 0.30 USD, which is over 10 times lower than it was 20 years ago (Fig. 1). This is commonly supported by the famous “Carlson Curves” that show trends in sequencing and synthesis costs over the years—biotechnology’s Moore’s law.[7] With this sophistication and commercialization of synthetic biology, “Anyone with a laptop computer can access public DNA sequence databases via the Internet, access free DNA design software, and place an order for synthesized DNA for delivery.”[5]

Figure 1: “Carlson Curve” highlighting the dramatic decrease in cost per base of synthetic DNA over the last 20 years. The yellow curve indicates the drop to around 0.3 USD/base pair of synthesized DNA.[7]

Synthetic DNA technologies have many purposes, ranging from making small changes in a known genetic sequence to the development and improvement of vaccines, like vaccinia for smallpox. The synthesis of viruses highlights the reason why synthetic biology is considered a “dual-use technology.” On the one hand, they offer hope in a wide variety of applications including global health and infectious disease. On the other, they could present potent threats to biosecurity if handled without caution or intentionally misused for destructive purposes.

Dual-use Technology

According to Matthew Meselson, a molecular biologist known for his discovery of DNA replication mechanisms:

“Every major technology—metallurgy, explosives, internal combustion, aviation, electronics, nuclear energy—has been intensively exploited, not only for peaceful purposes but also for hostile ones. Must this also happen with biotechnology, certain to be a dominant technology of the twenty-first century?”[8]

In the time since Meselson posed this question, the world has seen several instances suggesting that biotechnology will not be much different than its technological precursors. Perhaps most infamous instance came in September 2001, when letters containing Bacillus anthracis, a bacteria that causes the potentially fatal anthrax infection, were mailed to members of US Congress and media outlets just a week after the September 11 terrorist attacks.[6] Another case occurred in 2005 when the United States Center for Disease Control and Prevention (CDC) published their reconstruction of the influenza virus that caused the “Spanish Flu.” This particular viral epidemic had resulted in at least 50 million deaths worldwide in 1918, or 3-5% of the world’s population at the time.[6] The feat of synthesizing it in a modern day lab was impressive and alarming, since it demonstrated that lethal viruses that are, even those extinct from our world today, can be brought back and artificially synthesized using cutting-edge technology.

Letter sent to Senator Tom Daschle containing anthrax on October 15, 2001“Daschle letter” by Richard Taylor, licensed under public domain

Like the Spanish Flu, smallpox virus presents a prime example of dual-use research. Smallpox is an infectious and contagious disease characterized by high fevers and a progressive skin rash that is fatal in about three of ten people. After the last natural human outbreak in 1949, widespread vaccination against the variola major virus causing smallpox eventually led to the 1980 declaration of eradication by the 33rd World Health Assembly.[9] The variola major virus is now classified by the CDC as a Category A agent, alongside other viruses like anthrax that are the most easily transmitted and result in the highest mortality rates.[9] Variola major is only, supposedly, found in two locations—one lab in Russia and one in the United States—as designated by the World Health Organization. While there is hope that research on variola major will expand our understanding of complex virology and immunology, there remains the persistent threat that smallpox could be used as a biological weapon. For example, Ken Alibeck, former Deputy Director of the Soviet biological warfare agency, reported that the Soviet government had intentions to not only develop the variola major virus for use in biological weapons, but to produce “more virulent and contagious recombinant strains” of the virus.[10]

The vaccinia vaccine for smallpox is made from a virus similar to but less potent than the disease itself. Though routine vaccination in the United States ceased after 1972, the vaccine is stored in what is thought to be sufficient quantities to vaccinate between 6 and 7 million people.[10] Thus, if the next biological attack uses the variola major virus, we might be able to defend ourselves. But as indicated by Alibeck’s allegations, it is very conceivable, even likely, that the attack will come from something novel, perhaps related, but yet far more dangerous and less understood than the canonical smallpox disease.

Electron micrograph of the smallpox variola major virusImage by Dr. Fred Murphy, licensed under public domain

The development of unprecedented viruses or biological agents could happen as the result of dedicated, purposeful research, or it could happen by accident. Dr. David Relman, professor of Microbiology and Immunology at Stanford, notes that, “Kluging together different methods is the way molecular biology is most often done.” By “borrowing pieces from nature, sticking in synthetic pieces, and tweaking with genome editing tools,” scientists are pushing the boundaries of biology.[11] Occasionally, like in the 2001 case of an Australian research team working on a major pest control problem with mice, this can have unintended consequences, both positive and potentially negative.

In this study, researchers used standard genetic engineering techniques to insert the gene for interleukin-4 (IL-4) into the mousepox virus (a mouse-specific virus related to human smallpox) with the hopes of making infected mice infertile and thus curbing the population of the pests across Australia.[12] What they produced went far beyond expectations, not only sterilizing mice but killing them. The “super mousepox” virus was fatal even to mice that had already been successfully vaccinated against the original mousepox virus. The researchers were initially shocked and concerned by the treated mice’s ability to overcome vaccination, commenting that “this is the kind of thing that science fiction is made of.”[12]

This particular lethal gene used to modify the mousepox virus was specific to mice and possibly a few other rodent species, but could not cross over to primates. Thus the research itself is completely harmless to humans since it is incompatible with human genomes. However, the fact that these researchers were able to manipulate known sequences into something novel and impervious to our current vaccinations raised significant dual-use concerns. Cases like this mousepox study help guide the debate over how to best navigate the dual-use nature of synthetic biology from the standpoint of publishing information, regulating materials, and the incentives and education of scientists and the public alike.

Dual-use Threat, Multi-faceted Response

Information

Dr. Megan Palmer, senior research scholar at the Center for International Security and Cooperation (CISAC), surveyed approximately forty undergraduate bioengineering students at Stanford to ask how many of them thought that information should be controlled.[13] This would mean, for example, preventing the publication of sequences of pathogenic viruses. Not a single student raised their hand. Ronald Jackson and Ian Ramshaw, two of the primary researchers on the mousepox study, would agree. Even when faced with significant controversy and backlash after the publication of their vaccine-resistant virus, they insisted that the “worst thing we can do as scientists is try to hide what we are doing.”[12] Holding information from the public only further propagates suspicion and misunderstanding of science that could hinder scientific advancement. Even beyond that, the public plays a large role in helping to address dual-use issues and supporting research efforts. In order to facilitate this, we must continue to promote transparency of the advancements being made in the rapidly advancing field of genetic engineering.

Materials

If information cannot, and should not, be controlled, what about the materials themselves? Pathogenic sequences like those of the poxviruses can be easily accessed online, and with today’s technology, subsequently sequenced. Efforts to regulate synthetic biological material include the Biological Weapons Convention, an international treaty that is reviewed every five years in Geneva and now includes 156 States Parties and 16 unratified signatories. Domestic efforts strengthened in the aftermath of the September 11 terrorist attacks, with the creation of a list of Select Agents and Toxins that have the potential to threaten public health and safety.[14] This list was designed to regulate the transfer of genetic material within the United States, but has also become the fundamental basis by which commercial firms screen incoming orders for the synthesis of DNA.[5]

Legal control of materials is difficult to implement however, as was demonstrated by the IRTPA’s failure to effectively regulate the use of smallpox without also banning its vaccine. Legislation requires esoteric knowledge, like the fact that the vaccine for smallpox resides well within 85% sequence similarity as the smallpox virus itself. Once a law is put into place, changing or repealing it is an arduous process, and the field might move on before the law even has time to take effect. As David Relman cautions, “When you get into law, you’re using a very hard-edged, poorly controlled tool.”[11] However, there are options outside the strict confines of the legal system.

For example, in September of 2009, five (now eight) leading gene synthesis companies came together to form the International Gene Synthesis Consortium (IGSC). Currently accounting for approximately 80 percent of the world’s commercial gene synthesis, this organization aims to “safeguard biosecurity and promote the beneficial application of gene synthesis technology.”[15] Working with governments, the law, and science, they established screening procedures of both the sequences of ordered genes as well as the customers themselves to minimize the risk that their products be put to nefarious use. IGSC companies screen sequences of incoming orders according to the Select Agents and Toxins lists identified by the HHS, the USDA, and the Australia Group, an informal international coalition formed to control the spread of chemical and biological weapons.[16]

Dr. Terrence Tumpey examining a laboratory-grown reconstruction of the 1918 Spanish flu virus in a biosafety level 3 environment.“Influenza virus research” by James Gathany, licensed under public domain

When it comes to regulating synthetic biology, guidelines and regulatory agreements between like the IGSC between companies, research institutions, and even nations are likely to be less prone to error and easier to adapt as needed. Even if difficult to enforce, a set of more uniform and educated guidelines will establish a culture of safety and an expectation of adherence to international standards.

Incentive & Education

But rules and regulations alone will not be sufficient. As important actors in the field of synthetic biology, commercial firms can set a good example of one thing we should leverage—incentive. As expressed by the representatives of two gene synthesis companies, DNA2.0 and GENEART, firms “have the greatest incentive to ensure that the genes we synthesize do no harm and that the practice of gene synthesis remains safe”.[16] Even a small breach in the biosecurity of a company could damage the reputation and stability needed to facilitate research and scientific advancement.[16] Incentivizing development of standard guidelines and safety procedures, like in the commercial realm, could have a very positive influence on international biosecurity.

It seems indisputable that information must be shared, and in most cases, people can get their hands on dual-use materials if they so desire. What will unite and strengthen this field going forward are the attitudes and education of the people involved, from the students in bioengineering classes to the members of the Biological Weapons Conference to the commercial DNA synthesis firms.

It has been seventeen years since Matthew Meselson wondered how biotechnology would compare to its dual-use predecessors like aviation and nuclear energy. In those years, synthetic biology and related advancements have been utilized in both positive and negative ways. However, Meselson’s question has yet to be fully answered. And we, as developers, distributors, and consumers of this technology and its products, are capable of making sure it is answered in a way that preserves scientific advancement and discovery without endangering our the safety and security of our world.

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Fascinate is an online science publication run by undergraduate students at Stanford that focuses on communicating exciting scientific innovations in the fields of biology, physical sciences, health, technology, and more to the general public.